In biological tissues of a multicellular organism, various cells play their own roles and maintain harmonious functions as a whole. Alternatively, a part of the cells turn into cancer (herein, “cancer” and “tumor” are collectively referred to as “cancer”), then it becomes a neoplasm that is different from the surrounding region. There is not necessarily a clear-cut boundary between the cancerous region and a normal tissue region which is remote from the cancerous region, and the regions surrounding the cancerous region are also affected to some extent. Therefore, in order to analyze a function of cells in an organ tissue, it is necessary to separate and analyze a small number of cells present in a small region as simple as possible in a short period of time and with minimum loss.
In addition, in the field of regenerative medicine, organ stem cells are separated from a tissue and recultivated to induce differentiation in an attempt to regenerate the tissue of interest and eventually to regenerate the organ.
Cells need to be distinguished according to some sort of indicators in order to identify or separate the cell. In general, the following methods are used to distinguish cells.
1) Morphological cell sorting by visual observation: Examples include screening of bladder cancer or unitary tract cancer by assessing atypical cells that appear in the urine, as well as screening of cancers by sorting atypical cells in blood or cytologically diagnosing the tissue.2) Cell sorting by cell-surface antigen (marker) staining according to a fluorescence-antibody technique: A cell surface antigen, which is generally called a CD marker, is stained with a fluorescence-labeled antibody specific thereto. It is utilized for cell separation with a cell sorter, cancer screening using a flow cytometer or tissue staining, and the like. Indeed, they are not only used for medical care but also frequently used in physiological studies of cells and industrial application of cells.3) Alternatively, for separation of the stem cells, there is an example in which cells including stem cells are roughly separated using a fluorescent dye that is incorporated into the cells as a reporter, and subsequently stem cells of interest are separated by performing cultivation. In this case, since no effective marker of the stem cells has been established, the cells of interest are separated through cultivation using only those showing induction of differentiation.
It is an important technique for biological and medical analyses to separate and collect a particular cell(s) from a culture solution. When cells are to be separated based on the difference in the specific weight of the cells, they can be separated by velocity sedimentation. However, when there is little difference in their specific weights, such as when naive cells and sensitized cells are to be distinguished, cells need to be separated one by one based on information obtained from fluorescence-antibody staining or visual observation.
When it comes to such technique, there has been a cell sorter, for example. A cell sorter is the technique in which fluorescence-stained cells are isolated into an electrically-charged droplet on single-cell bases and allowed to fall as a drop. A high electrical field is applied in an arbitrary direction along the normal plane with respect to the falling direction of the drop while the drop is falling so as to control the destination of the falling drops based on the presence or absence of the fluorescence of the cells in the droplet and the magnitude of light scattering so as to fractionate the drops into multiple containers beneath and collect them (Non-patent Document 1: Kamarck, M. E., Methods Enzymol. Vol. 151, p 150-155 (1987)).
However, there are problems with this technique such as that it is expensive, the size of the apparatus is large, an electrical field as high as a few thousand volts is required, a large amount of sample that has been concentrated to a certain degree is required, the cells may be damaged during generation of the droplets, and the sample cannot directly be observed. In order to solve these problems, a cell sorter has recently been developed for which a micro manufacturing technique is used to prepare a fine channel so that cells that flow through a laminar flow in the channel can directly be observed with a microscope for separation (Non-patent Document 2: Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998); Non-patent Document 3: Analytical Chemistry, 70, pp. 1909-1915 (1998)). In a cell sorter produced by this micro manufacturing technique, however, the response speed for the separation of the sample is slow with respect to observation means. Thus, a need exists for a separation method that gives faster response and no damage to the sample. In addition, there are problems that the concentration of the cells in the sample solution under use has to be increased to a certain degree in advance or otherwise a subtle cell concentration prevents the separation efficiency of the device from being sufficiently enhanced; and further that when a small amount of sample is to be concentrated in a different apparatus, it is not only difficult to collect the concentrated solution without loss but also the cells might be contaminated during this cumbersome pretreatment stage, which are unfavorable in regenerative medicine and the like.
In order to solve these problems, the present inventors have utilized a micro manufacturing technique in developing a device for analyzing and separating a cell, which can be used to fractionate the sample based on the microconfiguration of the sample and the fluorescence distribution in the sample, and easily analyze and separate the cell sample without giving damage to the collected sample (Patent Document 1: Japanese Patent Unexamined Application Publication No. 2003-107099; Patent Document 2: Japanese Patent Unexamined Application Publication No. 2004-85323; Patent Document 3: WO2004/101731). This cell sorter is sufficiently practical in a laboratory level, but when it comes to general use in regenerative medicine, new techniques need to be developed for liquid transport process, collection process and pretreatments such as preparation of the sample.
Currently, although detection of cancerous tissues has been tremendously improved owing to the improvement in MRI (magnetic resonance imaging) and CT (computed tomography), no technique that is superior to the assessment method by biopsy for identifying benign and malignant tumors is present. There is one issue known for malignant tumors that the cancer cells metastasize from their tissues to other organs due to their ability to invade the blood vessels or the lymph vessels. Such malignant tumor cells that circulate in the peripheral blood are called peripheral blood-circulating cancer cells (Circulating Tumor Cells: CTCs), where approximately a few hundreds of cancer cells are thought to be present in one hundred thousand blood cells (including erythrocytes). Recently, carcinostatic agents for particular targets are being developed one after another, and it has become possible to select a carcinostatic drug that effectively disrupts the cell if the type of the malignant tumor in the blood is identified. If technique of monitoring CTCs flowing in the blood has been realized, the presence of malignant tumor cells causing the metastatic cancer flowing in the blood can quantitatively be measured. Then continuous and quantitative assessment of the effect of the administered carcinostatic drug will be possible, which will then lead to realization of the world's first technique that not only prevents administration of unnecessary carcinostatic drugs or administration of excessive amount of the carcinostatic drug, but also detects the presence or absence of recurrence.
With respect to genetic diagnosis and expression analysis, polymerase chain reaction (hereinafter, simply referred to as PCR) is a method for amplifying a particular nucleotide sequence from a mixture of various types of nucleic acids. Using PCR, a particular nucleic acid sequence can be amplified by adding a DNA template such as genomic DNA or complementary DNA that has been reverse-transcribed from messenger RNA, two or more types of primers, a thermostable enzyme, salt such as magnesium, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) to a mixture of various types of nucleic acids, and then repeating the following steps at least one cycle: a step of separating the nucleic acids into single strands; a step of binding the primers with the separated nucleic acids; and a step of using the primer-bound nucleic acids as a template for hybridization using the thermostable enzyme. According to PCR, a thermal cycle is utilized in which the temperature of a reaction container used for the DNA amplification reaction is increased and decreased. Various mechanisms are used for changing the temperature. Examples include: a mechanism in which the temperature of a reaction container containing a sample is changed by heat exchange using a heater, a Peltier device or hot air; a mechanism in which the temperature is changed by alternately bringing a reaction container into contact with block heaters and liquid vessels at different temperatures; and a mechanism in which the temperature is changed by running a sample in a channel that has regions with different temperatures. An example of a currently commercially available device with the highest speed includes LightCycler from Roche. LightCycler has a system in which a sample, DNA polymerase, DNA fragments as primers and a fluorescence-labeling dye for measurement are introduced into each of a plurality of glass capillary tubes, and the temperature of a minute amount of the droplet in this capillary tube is changed by blowing hot air at the same temperatures as the temperatures required for the droplet to be changed (for example, between two temperatures of 55° C. and 95° C.), and at the same time, irradiating this glass capillary tube with excitation light for the fluorescent dye, thereby allowing measurement of the obtained fluorescence intensity.
The temperature of a sample can be changed repeatedly by these methods.